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Gene activation-associated long noncoding RNAs function in mouse preimplantation development.

Hamazaki N, Uesaka M, Nakashima K, Agata K, Imamura T - Development (2015)

Bottom Line: Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes.Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation.Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Global COE Program, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan Division of Basic Stem Cell Biology, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

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Effect of pancIl17d knockdown on colony outgrowth from blastocysts and on ESC properties. (A) Rate of colony outgrowth from knockdown and rescued blastocysts. Colonies growing after 10 days in culture were counted. (B) Representative images of colonies derived from siRNA-injected embryos. (C) Box plot of diameter of colonies derived from pancRNA knockdown blastocysts. (D) Number of ESCs 24 h after siRNA introduction by electroporation. (E) Proportion of apoptotic cells detected by TUNEL staining in knocked down ESCs after siRNA introduction. (F) Proportion of proliferating ESCs, as analyzed by EdU labeling. (G) Expression levels of pancIl17d, Il17d and pluripotency marker genes in ESCs, as detected by RT-qPCR. Gapdh was used as a control. The expression level in control-transfected ESCs was set as 1. Asterisks indicate significant differences compared with si Control samples. *P<0.05; **P<0.01; ***P<0.001. Error bars indicate s.e.m.
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DEV116996F5: Effect of pancIl17d knockdown on colony outgrowth from blastocysts and on ESC properties. (A) Rate of colony outgrowth from knockdown and rescued blastocysts. Colonies growing after 10 days in culture were counted. (B) Representative images of colonies derived from siRNA-injected embryos. (C) Box plot of diameter of colonies derived from pancRNA knockdown blastocysts. (D) Number of ESCs 24 h after siRNA introduction by electroporation. (E) Proportion of apoptotic cells detected by TUNEL staining in knocked down ESCs after siRNA introduction. (F) Proportion of proliferating ESCs, as analyzed by EdU labeling. (G) Expression levels of pancIl17d, Il17d and pluripotency marker genes in ESCs, as detected by RT-qPCR. Gapdh was used as a control. The expression level in control-transfected ESCs was set as 1. Asterisks indicate significant differences compared with si Control samples. *P<0.05; **P<0.01; ***P<0.001. Error bars indicate s.e.m.

Mentions: To further investigate the significance of pancIl17d for embryonic development, we plated the surviving pancIl17d knockdown blastocysts in medium containing mouse LIF and inhibitors for MEK1/2 (MAP2K1/2) and GSK3β (2i medium), conditions that are frequently utilized for the culture of ground-state ESCs, and harvested the cultures after 10 days. Whereas about 70% of the control siRNA-injected embryos produced ESC-like colonies on average, only 10-20% of the pancIl17d knockdown embryos did so (Fig. 5A). Even when pancIl17d knockdown embryos did produce colonies, they were significantly smaller than those derived from control siRNA-injected embryos (Fig. 5B,C), indicating that pancIl17d knockdown decreases the ability to form a colony. These knockdown-induced impairments were also rescued by the addition of rIL17D to the culture medium at the 4-cell stage, strongly suggesting that the effects of pancIl17d knockdown are mediated by the downregulation of Il17d gene expression.Fig. 5.


Gene activation-associated long noncoding RNAs function in mouse preimplantation development.

Hamazaki N, Uesaka M, Nakashima K, Agata K, Imamura T - Development (2015)

Effect of pancIl17d knockdown on colony outgrowth from blastocysts and on ESC properties. (A) Rate of colony outgrowth from knockdown and rescued blastocysts. Colonies growing after 10 days in culture were counted. (B) Representative images of colonies derived from siRNA-injected embryos. (C) Box plot of diameter of colonies derived from pancRNA knockdown blastocysts. (D) Number of ESCs 24 h after siRNA introduction by electroporation. (E) Proportion of apoptotic cells detected by TUNEL staining in knocked down ESCs after siRNA introduction. (F) Proportion of proliferating ESCs, as analyzed by EdU labeling. (G) Expression levels of pancIl17d, Il17d and pluripotency marker genes in ESCs, as detected by RT-qPCR. Gapdh was used as a control. The expression level in control-transfected ESCs was set as 1. Asterisks indicate significant differences compared with si Control samples. *P<0.05; **P<0.01; ***P<0.001. Error bars indicate s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4352986&req=5

DEV116996F5: Effect of pancIl17d knockdown on colony outgrowth from blastocysts and on ESC properties. (A) Rate of colony outgrowth from knockdown and rescued blastocysts. Colonies growing after 10 days in culture were counted. (B) Representative images of colonies derived from siRNA-injected embryos. (C) Box plot of diameter of colonies derived from pancRNA knockdown blastocysts. (D) Number of ESCs 24 h after siRNA introduction by electroporation. (E) Proportion of apoptotic cells detected by TUNEL staining in knocked down ESCs after siRNA introduction. (F) Proportion of proliferating ESCs, as analyzed by EdU labeling. (G) Expression levels of pancIl17d, Il17d and pluripotency marker genes in ESCs, as detected by RT-qPCR. Gapdh was used as a control. The expression level in control-transfected ESCs was set as 1. Asterisks indicate significant differences compared with si Control samples. *P<0.05; **P<0.01; ***P<0.001. Error bars indicate s.e.m.
Mentions: To further investigate the significance of pancIl17d for embryonic development, we plated the surviving pancIl17d knockdown blastocysts in medium containing mouse LIF and inhibitors for MEK1/2 (MAP2K1/2) and GSK3β (2i medium), conditions that are frequently utilized for the culture of ground-state ESCs, and harvested the cultures after 10 days. Whereas about 70% of the control siRNA-injected embryos produced ESC-like colonies on average, only 10-20% of the pancIl17d knockdown embryos did so (Fig. 5A). Even when pancIl17d knockdown embryos did produce colonies, they were significantly smaller than those derived from control siRNA-injected embryos (Fig. 5B,C), indicating that pancIl17d knockdown decreases the ability to form a colony. These knockdown-induced impairments were also rescued by the addition of rIL17D to the culture medium at the 4-cell stage, strongly suggesting that the effects of pancIl17d knockdown are mediated by the downregulation of Il17d gene expression.Fig. 5.

Bottom Line: Expression of these bidirectional promoter-associated noncoding RNAs (pancRNAs) was strongly associated with the upregulation of their cognate genes.Conversely, knockdown of three abundant pancRNAs led to reduced mRNA expression, accompanied by sustained DNA methylation even in the presence of enzymes responsible for DNA demethylation.Thus, this novel class of lncRNAs can modulate the transcription machinery in cis to activate zygotic genes and is important for preimplantation development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics and Global COE Program, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan Division of Basic Stem Cell Biology, Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

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Related in: MedlinePlus